For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines. In our current studies a strain of Escherichia coli was constructed that contained deletions in all of the genes involved in polyamine biosynthesis;namely, speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase). Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free media;albeit at a slightly (ca 40-50%) reduced growth rate. These results are of particular significance since they show that good growth can take place in the complete absence of any polyamines. This finding is surprising since there are so many reports in the literature (mainly based on in vitro studies) reporting polyamine requirements for protein synthesis, DNA and ribosome structure, etc. In these studies we also found that even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, surprisingly this strain showed a requirement for polyamines for growth under strictly anaerobic conditions. Our current studies in E. coli are also concerned with glutathionylspermidine. As we have previously shown, all of the spermidine of the E. coli cells and a large percentage of the intracellular glutathione are converted to glutathionylspermidine at the end of the logarithmic growth. Nothing is known about the function of glutathionylspermidine in E. coli, although it has been shown to of critical importance in trypanosomes. Our current studies aim at finding the physiological function of glutathionylspermidine in E. coli. A mutant of E. coli is available that lacks the gene for glutathionylspermidine synthetase/ (gsp). We have compared the phenotypic effects of the gsp gene deletion in E. coli with a wild type strain in various growth conditions (air, 95% oxygen, temperature and copper toxicity) and found no difference between the mutant and the wild type. However, microarray studies show several differences between the mutant and the wild type strains. The mutant show more than 4-fold increase in expression of zraP (zinc resistance protein), copA (copper transporter) and more than 2-fold increase in cueO (multicopper oxidase), cusF (periplasmic copper binding protein). Protein comparisons (in 2D arrays) of the two strains are underway.